Plasminogen activators are widely used in the treatment of thromboembolic diseases. One of these activators is urokinase plasminogen activator (uPA), known to be synthesized as a proenzyrne consisting of a single-chain protein (scuPA) [Pannell, R. & Gurewich, V., Blood 69:22-28 (1987)].
Limited proteolysis of scuPA results in the formation of two chains (tcuPA), considered to be the active form of the enzyme [Kasai. S., et al. J. Biol. Chem. 260:12382-12389 (1985)]. One of the most important regulators of uPA is the plasminogen activator inhibitor-1 (PAI-1). This regulator interacts with tcuPA in a very rapid two-step reaction, leading to the formation of an inactive, SDS-stable 1/1 complex [Lindahl, T. L., et al., Biochem. J. 265:109-113 (1990)]. The binding of tcuPA to UPAR only slightly reduces the susceptibility of tcuPA to the inhibitory effect of PAI-1 [Ellis, V., etal., J. Biol. Chem. 265:9904-9908 (1990)].
More recent reports by the inventor have shown that the proenzyme (scuPA) can be activated by an alternative mechanism. Binding of scuPA to a soluble form of the urokinase plasminogen activator receptor (suPAR) leads to activation of the enzyme without prior cleavage [Higazi, A A-R., et al., J. Biol. Chem. 270:17375-17380 (1995)]. Single-chain urokinase plasminogen activator (scuPA) is the unique form of urokinase secreted by cells. ScuPA has low intrinsic activity, but cleavage of a single peptide bond leads to the formation of the active, two-chain urokinase plasminogen activator (tcuPA). The activity of the scuPA/suPAR complex is relatively resistant to PAI-1. The mechanism of PAI-1 inhibition of the scuPA/suPAR complex is competitive and reversible [Higazi, A A-R., et al., Blood 87:3545-3549 (1996)]. Resistance of the scuPA/suPAR complex to other inhibitors, such as PAI-2, has also been described [Schwartz, B. S., J. Biol. Chem. 269:8319-8323 (1994)]. Evidently, when the two forms of an enzyme are fully active, the susceptibility to the effects of regulators present in the physiological environment will determine which is the active or “more active” form. The fact that the activity of the scuPA/suPAR complex is similar to that of tcuPA, whereas its sensitivity to inhibition is lower, led to the finding that under physiological conditions the complex is the active form of urokinase, which underlies the present invention.
The opinion was that tcuPA is the physiological urokinase plasminogen activator whereas scuPA serves as the proenzyme. This concept was based on the greater catalytic activity of tcuPA, as compared to scuPA. To the contrary, more recent studies have provided evidence that the activity of scuPA, bound to its receptor, suPAR, is in the same range as that of tcuPA [Higazi, A A-R., (1995) ibid.; Higazi, A A-R. et al. (1996) ibid.]. On the other hand, Behrendt et al. and Ellis et al. [Behrendt N., et al., Biol. Chem. 376:259-279 (1995); Ellis V., J. Biol. Chem. 271:14779-14784 (1996)] described suPAR as having no stimulatory effect on scuPA activity. However, a more recent publication [Behrendt N. & Dano K, FEBS Lett. 339:31-36 (1996)] describes a stimulatory effect of suPAR on scuPA activity, but the mechanism of this stimulation was questioned [Higazi, A A-R, FEBS Lett 402:291-292 (1997)].
Some of the conflicting results probably stemmed from different experimental conditions and chromogenic substrates of plasmin. The inventor found that when using the physiological substrate of the fibrinolytic system, i.e. human plasma clots, suPAR stimulated the intrinsic activity of scuPA. Using a plasma-derived clot as a substrate of the fibrinolytic system, the activity of the scuPA/suPAR complex was significantly greater than that of tcuPA. On the other hand, suPAR inhibited the scuPA-mediated fibrinolytic activity when the clot was prepared with purified fibrinogen. Addition of serum to purified fibrinogen resulted in stimulation of suPAR/scuPA-mediated fibrinolysis. It was therefore concluded that activation or inhibition of scuPA activity by suPAR depends on the substrate used. For example, with H-D-norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide D-lactate salt (Spect PL), suPAR stimulated scuPA activity, whercas suPAR was found to inhibit scuPA-mediated plasminogen activation when the activin, of the generated plasmin was determined with a synthetic plasmin substrate H-D-val-leu-lys-p-nitroanilide (S-2251) [Higazi, A A-R., Thromb. Res. 84:243-252 (1996)]. As noted above, it has been suggested that under physiological conditions the complex is the active form of urokinase. Since it appeared that the regulation of scuPA activity by suPAR depends on the nature of the substrate used, the inventor proceeded to examine the suitability of the physiological substrate of the fibrinolytic system, i.e. the plasma clot.
As will be shown hereinafter, suPAR stimulated the activity mediated by scuPA on a clot generated by the coagulation of human plasma. Using a plasma-derived clot as substrate of the fibrinolytic system, the activity of the scuPA/suPAR complex was significantly greater than that of tcuPA. It will further be shown that the effect of serum depends on the presence of intact suPAR. Optimal stimulation of scuPA/suPAR-mediated fibrinolysis will be obtained when serun is incorporated into a clot during the coagulation process.
Therefore, one aim of the present invention is to provide a scuPA/suPAR complex that acts as a plasminogen activator in combination or in the presence of a specific factor that stimulates the fibrinolytic activity of the scuPA/suPAR complex. While searching for such specific factor, the inventor has surprisingly found that it is human IgG protein or an IgG derived peptidewhich have the stimulating effect on the activity of the complex.